The translocation of proteins into and across membranes is an essential step in organelle biogenesis and protein topogenesis. The goal of this project is to understand the molecular mechanisms by which protein are integrated into or across membranes. The experiments focus on the regulation of protein-conducting channels that are observed in endoplasmic reticulum. The specific aims are to understand the regulation of the gating of this channel and to identify, purify and reconstitute the channel and its regulatory proteins. There are three steps of regulation that will be studied: What opens the channel to allow proteins to move across the membrane; what closes it after a protein has moved across; and, when it integrates a transmembrane protein, what opens the channel to the lipid bilayer thereby allowing displacement of latent transmembrane domains from the channel's aqueous environment into the hydrophobic milieu of the bilayer. The experimental approach is a hybrid of two techniques: reconstitution of membrane proteins into planar lipid bilayers and the reconstitution of protein translocation across membranes. Protein translocation is a membrane transport process. Planar lipid bilayers are optimal for studying such processes since they allow both complete control over the lipid and protein components as well as access for experimental manipulation and electrophysiological tools to both sides of the membrane. These tools are used to assay the status (open-close) of the channel. Three approaches are being used for identification and purification of the protein-conducting channels. (i) Protein translocation can be reconstituted after detergent solubilization of either pancreatic endoplasmic reticulum rough microsomes and E. coli inverted plasma membrane vesicles. Proteins can be fractionated prior to dialysis of the detergent to identify those necessary for protein translocation. (ii) Ribosomes are being used to purify integral membrane proteins of the endoplasmic reticulum that are linked to the ribosome via a nascent peptide chain. (iii) Proteins that are known to be essential for protein translocation (such as the six transmembrane proteins of the mammalian ER signal peptidase complex, yeast SEC 62, mitochondrial import receptors, E. coli prlA) are being purified and reconstituted into proteoliposomes to test if any of these (or a combination of these) are sufficient for activity of the protein-conducting channel. These are the first protein-conducting channels to be described. Similar techniques are being applied to the study of other "signal sequence" mediated protein translocations (the E. coli inner membrane, mitochondria, chloroplast). The goal is to identify general principles that may regulate protein topogenesis and organelle biogenesis.